US8609405B2 - GABA neuron progenitor cell marker 65B13 - Google Patents

GABA neuron progenitor cell marker 65B13 Download PDF

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US8609405B2
US8609405B2 US12/524,153 US52415308A US8609405B2 US 8609405 B2 US8609405 B2 US 8609405B2 US 52415308 A US52415308 A US 52415308A US 8609405 B2 US8609405 B2 US 8609405B2
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gaba
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neuron progenitor
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Yuichi Ono
Yasuko Nakagawa
Eri Mizuhara
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Eisai R&D Management Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5073—Stem cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9406—Neurotransmitters
    • G01N33/9426—GABA, i.e. gamma-amino-butyrate

Definitions

  • the present invention provides a 65B13 gene as a marker for GABA neuron progenitor cells, and relates to the use of the gene in identifying GABA neuron progenitor cells.
  • the brain functions by forming a complex network from a great variety of neurons. Its failure may result in various neurological diseases.
  • transplantation and regeneration therapies are currently investigated. The most important thing in these therapeutic methods is to correctly identify various types of neurons in transplantation materials. Furthermore, from the viewpoint of improvement of safety and therapeutic effect, it is desirable to isolate only the type of cells that are needed for transplantation.
  • the cerebellum works on smooth motor functions such as regulation of balance, posture, and voluntary movement.
  • Functional recovery can be achieved by replenishing lost neurons and reconstituting the network.
  • dorsal horn there is an area called “dorsal horn” in the dorsal spinal cord.
  • Dorsal root ganglion neurons that detect stimuli from the periphery transmit signals to the dorsal horn interneurons, and the signals are further transmitted to the brain.
  • the dorsal horn contains excitatory glutamatergic neurons and inhibitory GABA neurons. The balance between the two adequately regulates the signal transmission. Inactivation of GABA neurons results in chronic pain, etc.
  • the 65B13 gene is known to be transiently expressed in dopamine-producing neuron progenitor cells after the termination of cell division (see Patent Document 1); however, there is no report published on the connection between the gene and GABA neuron. Furthermore, it has been reported that the types of spinal cord interneurons and Purkinje cells can be identified by using the expression of the Corl1 or Corl2 gene as an indicator, respectively (see Patent Documents 2 and 3). However, to date there is no known marker that can selectively identify GABA neuron progenitor cells.
  • the transcription factor Ptf1a is known to be expressed in GABA progenitor cells; however, it is a transcription factor, and there is no known membrane protein that is useful as a selection marker (Non-Patent Documents 1 and 2).
  • An objective of the present invention is to provide markers that enable selective identification of GABA neuron progenitor cells.
  • Another objective of the present invention is to provide methods of using the markers as an indicator to identify GABA neuron progenitor cells, and reagents for use in these methods.
  • the present inventors identified a selective marker, 65B13, for GABA neuron progenitor cells in the spinal dorsal horn and cerebellum, and successfully isolated GABA neuron progenitor cells using antibodies that bind to the protein encoded by the gene. Specifically, 65B13 was demonstrated to be useful as a marker to isolate GABA-producing neuron progenitor cells in the spinal dorsal horn and cerebellum.
  • GABA neuron progenitor cells can be efficiently identified or isolated by using the marker identified by the present inventors as an indicator.
  • the present invention relates to markers that enable selective identification of GABA neuron progenitor cells, methods of using the markers as an indicator to identify GABA neuron progenitor cells, and reagents for use in the methods. More specifically, the present invention provides:
  • FIG. 1 shows the expression pattern of the 65B13 gene in the nervous system of fetal mouse.
  • FIG. 2A shows a developmental scheme for the dorsal spinal cord.
  • FIG. 2B shows identification markers for various neurons.
  • FIG. 3 shows a comparison of the 65B13, Mash1, and Pax2 expressions in the spinal cord of E10.5 mouse.
  • FIG. 4 shows a comparison of the 65B13, Corl1, and Mash1 expressions in the spinal cord of E12.5 mouse.
  • FIG. 5 shows differentiation of 65B13-positive cells isolated from the spinal cord of E12.5 mouse into GABA-producing dILA neurons.
  • FIG. 5A shows a result of flow cytometry analysis using an anti-65B13 antibody;
  • FIG. 5B shows marker staining of the isolated 65B13-positive cells after two days of culture.
  • FIG. 6 shows a comparison of the 65B13, Corl2, and Pax2 expressions in the fetal cerebellar primordium.
  • FIG. 6A shows the result of expression analysis at E12.5;
  • FIG. 6B shows the result of expression analysis at E14.5.
  • the left and right photographs show the expression of 65B13 or Pax2, respectively.
  • FIG. 7 shows that 65B13-positive cells could be isolated from the cerebellar primordia of fetal mouse.
  • FIG. 7A shows results for the cerebella and 65B13-positive cells isolated from the E12.5 cerebella.
  • FIG. 7B shows results of the cerebella and 65B13-positive cells isolated from the E14.5 cerebella.
  • FIG. 8 shows differentiation of 65B13-positive cells isolated from the cerebellar primordia of E12.5 (A) and E14.5 (B) mice into GABA-producing Purkinje cells and GABA-producing Golgi cells, respectively.
  • FIG. 9 shows 65B13-positive cells could be isolated from a population of in vitro differentiated spinal cord neurons derived from ES cells and their differentiation into GABA-producing neurons.
  • FIG. 10 shows the structures of DNA constructs that can be used to select GABA-producing neuron progenitor cells.
  • FIG. 11 shows that a foreign gene (Gsh1) could be expressed specifically in GABA-producing neuron progenitor cells by using the 65B13 promoter.
  • the present inventors demonstrated that the 65B13 gene was selectively expressed in GABA neuron progenitor cells of spinal dorsal horn and cerebellum.
  • the present invention provides polynucleotides for detecting or selecting GABA-producing neuron progenitor cells.
  • the 65B13 gene of the present invention includes, for example, two types of genes named 65B13-a and 65B13-b, which are alternative isoforms of the 65B13 gene.
  • the respective nucleotide sequences are shown in SEQ ID NOs: 1 and 3, and the amino acid sequences encoded by the genes are shown in SEQ ID NOs: 2 and 4.
  • the coding region of 65B13-a starts with A at position 178 in SEQ ID NO: 1, and extends to the stop codon at positions 2278 to 2280, encoding a protein of 700 amino acids.
  • the 17 amino acid residues encoded by the sequence of positions 178 to 228 constitute a signal sequence, while the 17 amino acid residues encoded by the sequence of positions 1717 to 1767 constitute a transmembrane region.
  • the coding region of 65B13-b starts with A at position 127 in SEQ ID NO: 2 and extends to the stop codon at positions 2277 to 2079, encoding a protein of 650 amino acids.
  • the 17 amino acid residues encoded by the sequence of positions 127 to 178 constitute a signal sequence, while the 17 amino acid residues encoded by the sequence of positions 1516 to 1566 constitute a transmembrane region.
  • the 65B13 gene of the present invention also includes, for example, the genes of SEQ ID NOs: 33, 35, 37, 39, 41, 43, 45, 47, and 49.
  • the amino acid sequences encoded by the genes are shown in SEQ ID NOs: 34, 36, 38, 40, 42, 44, 46, 48, and 50, respectively.
  • the coding region of SEQ ID NO: 33 starts with A at position 668 and extends to the stop codon at positions 2768 to 2770, encoding a protein of 700 amino acids.
  • the 19 amino acid residues encoded by the sequence of positions 668 to 724 constitute a signal sequence, while the 494 amino acid residues encoded by the sequence of positions 725 to 2206 constitute an extracellular domain.
  • the coding region of SEQ ID NO: 35 starts with A at position 130 and extends to the stop codon at positions 2230 to 2232, encoding a protein of 700 amino acids.
  • the 19 amino acid residues encoded by the sequence of positions 130 to 186 constitute a signal sequence, while the 494 amino acid residues encoded by the sequence of positions 187 to 1668 constitute an extracellular domain.
  • the coding region of SEQ ID NO: 37 starts with A at position 199 and extends to the stop codon at positions 2098 to 2100, encoding a protein of 633 amino acids.
  • the 20 amino acid residues encoded by the sequence of positions 199 to 258 constitute a signal sequence, while the 490 amino acid residues encoded by the sequence of positions 259 to 1728 constitute an extracellular domain.
  • the coding region of SEQ ID NO: 39 starts with A at position 199 and extends to the stop codon at positions 2323 to 2325, encoding a protein of 708 amino acids.
  • the 20 amino acid residues encoded by the sequence of positions 199 to 258 constitute a signal sequence, while the 490 amino acid residues encoded by the sequence of positions 259 to 1728 constitute an extracellular domain.
  • the coding region of SEQ ID NO: 41 starts with A at position 199 and extends to the stop codon at positions 2323 to 2325, encoding a protein of 708 amino acids.
  • the 20 amino acid residues encoded by the sequence of positions 199 to 258 constitute a signal sequence, while the 490 amino acid residues encoded by the sequence of positions 259 to 1728 constitute an extracellular domain.
  • the coding region of SEQ ID NO: 43 starts with A at position 15 and extends to the stop codon at positions 1914 to 1916, encoding a protein of 633 amino acids.
  • the 20 amino acid residues encoded by the sequence of positions 15 to 74 constitute a signal sequence, while the 490 amino acid residues encoded by the sequence of positions 75 to 1544 constitute an extracellular domain.
  • the coding region of SEQ ID NO: 45 starts with A at position 199 and extends to the stop codon at positions 1948 to 1950, encoding a protein of 583 amino acids.
  • the 20 amino acid residues encoded by the sequence of positions 199 to 258 constitute a signal sequence, while the 440 amino acid residues encoded by the sequence of positions 259 to 1578 constitute an extracellular domain.
  • the coding region of SEQ ID NO: 47 starts with A at position 15 and extends to the stop codon at positions 1764 to 1766, encoding a protein of 583 amino acids.
  • the 20 amino acid residues encoded by the sequence of positions 15 to 74 constitute a signal sequence, while the 440 amino acid residues encoded by the sequence of positions 75 to 1394 constitute an extracellular domain.
  • the coding region of SEQ ID NO: 49 starts with A at position 196 and extends to the stop codon at positions 2260 to 2262, encoding a protein of 688 amino acids.
  • the 20 amino acid residues encoded by the sequence of positions 196 to 255 constitute a signal sequence, while the 470 amino acid residues encoded by the sequence of positions 256 to 1665 constitute an extracellular domain.
  • the 65B13 gene of the present invention also includes, for example, the sequences of accession numbers XM — 994164, AL136654, XM — 512603, XR — 012248, XM — 541684, and XM — 583222.
  • the polynucleotides of the present invention include polynucleotides for detecting or selecting GABA-producing neuron progenitor cells, which can hybridize to a nucleotide selected from (i), (ii), (iii), and (iv) below, or to a complementary sequence thereof.
  • a “polynucleotide” refers to a polymer comprising nucleotides or nucleotide pairs of multiple deoxyribonucleic acids (DNA) or ribonucleic acids (RNA), and includes DNA, cDNA, genomic DNA, chemically synthesized DNA, and RNA.
  • polynucleotides can also contain non-naturally occurring nucleotides such as 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 2′-O-methylcytidine, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, dihydrouridine, 2′-O-methylpseudouridine, ⁇ -D-galactosylqueuosine, 2′-O-methylguanosine, inosine, N6-isopentenyladenosine, 1-methyladenosine, 1-methylpseudouridine, 1-methylguanosine, 1-methylinosine, 2,2-dimethylguanosine, 2-methyladenosine, 2-methylguanosine, 3-methylcytidine, 5-methylcytidine, N6-methyladenosine, 7-methylguanosine, 5-methylaminomethyluridine, 5-methoxyaminomethyl-2-
  • polynucleotides of the present invention can also be produced by chemical synthesis based on the known sequence of 65B13. Alternatively, such polynucleotides can be prepared from 65B13 gene-expressing cells using hybridization, PCR, etc.
  • a target protein has the same biological property as a 65B13 protein (for example, the amino acid sequence of SEQ ID NO: 2, 4, 34, 36, 38, 40, 42, 44, 46, 48, or 50).
  • the biological property of a 65B13 protein includes, for example, the olfactory nerve network pattern.
  • the selective expression in GABA neuron progenitor cells can also be regarded as a function (biological property).
  • a target protein has the equivalent biological property as the amino acid sequence of SEQ ID NO: 2, 4, 34, 36, 38, 40, 42, 44, 46, 48, or 50 identified by the present inventors can be assessed by those skilled in the art, for example, by testing for the olfactory nerve network pattern or selective expression in GABA neuron progenitor cells.
  • polynucleotides of the present invention also include polynucleotides that hybridize under stringent conditions to a polynucleotide comprising the nucleotide sequence of positions 178 to 2280 in SEQ ID NO: 1, the nucleotide sequence of positions 127 to 2079 in SEQ ID NO: 3, the nucleotide sequence of positions 668 to 2770 in SEQ ID NO: 33, the nucleotide sequence of positions 130 to 2232 in SEQ ID NO: 35, the nucleotide sequence of positions 199 to 2100 in SEQ ID NO: 37, the nucleotide sequence of positions 199 to 2325 in SEQ ID NO: 39, the nucleotide sequence of positions 15 to 2325 in SEQ ID NO: 41, the nucleotide sequence of positions 15 to 1916 in SEQ ID NO: 43, the nucleotide sequence of positions 199 to 1950 in SEQ ID NO: 45, the nucleotide sequence of positions 15 to 1766 in SEQ ID NO: 47, or the nu
  • polynucleotides can be obtained from cDNA libraries and genomic libraries of animals such as humans, mice, rats, rabbits, hamsters, chickens, pigs, cows, goats, sheep, monkeys, and dogs by known hybridization methods such as colony hybridization, plaque hybridization, and Southern blotting using as a probe a polynucleotide comprising the nucleotide sequence of positions 1 to 2876 in SEQ ID NO: 1, the nucleotide sequence of positions 1 to 2243 in SEQ ID NO: 3, the nucleotide sequence of positions 1 to 3123 in SEQ ID NO: 33, the nucleotide sequence of positions 1 to 3247 in SEQ ID NO: 35, the nucleotide sequence of positions 1 to 2153 in SEQ ID NO: 37, the nucleotide sequence of positions 1 to 2979 in SEQ ID NO: 39, the nucleotide sequence of positions 1 to 2973 in SEQ ID NO: 41, the nucleotide sequence of positions 1 to 1969 in SEQ ID
  • total RNA is first prepared from cells, organs, tissues, or such that express a polynucleotide of the present invention, by known techniques such as guanidine ultracentrifugation (Chirwin et al. Biochemistry 1979, 18:5294-5299) or AGPC (Chomczynski and Sacchi Anal. Biochem. 1987, 162:156-159), followed by mRNA purification using an mRNA purification kit (Pharmacia), etc.
  • a kit for direct mRNA preparation such as the QuickPrep mRNA Purification Kit (Pharmacia), may also be used.
  • cDNA is synthesized from the resulting mRNA using reverse transcriptase.
  • cDNA synthesis kits such as the AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Corporation) are also available commercially. Other methods that use the 5′-RACE method to synthesize and amplify cDNA by PCR may also be used (Frohman et al. Proc. Natl. Acad. Sci. USA 1988, 85:8998-9002; Belyaysky et al. Nucleic Acids Res. 1989, 17:2919-32). In addition, in order to construct cDNA libraries containing a high percentage of full-length clones, known techniques such as the oligo-capping method (Maruyama and Sugano. Gene 1994, 138:171-4; Suzuki. Gene 1997, 200:149-56) can also be employed. The cDNA obtained in this manner is then incorporated into a suitable vector.
  • hybridization conditions in the present invention include “2 ⁇ SSC, 0.1% SDS, 50° C.”, “2 ⁇ SSC, 0.1% SDS, 42° C.”, and “1 ⁇ SSC, 0.1% SDS, 37° C.”.
  • conditions of higher stringency include “2 ⁇ SSC, 0.1% SDS, 65° C.”, “0.5 ⁇ SSC, 0.1% SDS, 42° C.”, and “0.2 ⁇ SSC, 0.1% SDS, 65° C.”. More specifically, a method that uses the Rapid-hyb buffer (Amersham Life Science) can be carried out by performing pre-hybridization at 68° C. for 30 minutes or more, adding a probe to allow hybrid formation at 68° C.
  • conditions of higher stringency can be achieved by increasing the temperature for pre-hybridization, hybridization, or second wash.
  • the pre-hybridization and hybridization temperature can be raised to 60° C., and to 68° C. for higher stringency.
  • a person with ordinary skill in the art can also integrate other conditions such as probe concentration, probe length, and reaction time, to obtain isoforms of 65B13 of the present invention, allelic mutants, and corresponding genes derived from other organisms.
  • hybridizing polynucleotides include polynucleotides containing a nucleotide sequence that has at least 50% or more, preferably 70%, more preferably 80%, and even more preferably 90% (for example, 95% or more, or 99%) identity with a nucleotide sequence comprising the nucleotide sequence of positions 178 to 2280 in SEQ ID NO: 1, the nucleotide sequence of positions 127 to 2079 in SEQ ID NO: 3, the nucleotide sequence of positions 668 to 2770 in SEQ ID NO: 33, the nucleotide sequence of positions 130 to 2232 in SEQ ID NO: 35, the nucleotide sequence of positions 199 to 2100 in SEQ ID NO: 37, the nucleotide sequence of positions 199 to 2325 in SEQ ID NO: 39, the nucleotide sequence of positions 15 to 2325 in SEQ ID NO: 41, the nucleotide sequence of positions 15 to 1916 in SEQ ID NO: 43, the nucleotide sequence
  • Such identities can be determined by the BLAST algorithm (Altschul. Proc. Natl. Acad. Sci. USA 1990, 87:2264-8; Karlin and Altschul. Proc. Natl. Acad. Sci. USA 1993, 90:5873-7).
  • Examples of programs that have been developed based on this algorithm include the BLASTX program for determining the identity of amino acid sequences, and the BLASTN program for nucleotide sequences (Altschul et al. J. Mol. Biol. 1990, 215:403-10). These programs can be used for the sequences of the present invention.
  • One can refer to, for example, http://www.ncbi.nlm.nih.gov for a specific example of analysis methods.
  • the 65B13 isoforms or allelic mutants, and other genes with a 65B13-like structure and function can be obtained from cDNA libraries and genome libraries of animals such as humans, mice, rats, rabbits, hamsters, chickens, pigs, cows, goats, sheep, monkeys, and dogs by designing primers based on the nucleotide sequence of positions 178 to 2280 in SEQ ID NO: 1, the nucleotide sequence of positions 127 to 2079 in SEQ ID NO: 3, the nucleotide sequence of positions 668 to 2770 in SEQ ID NO: 33, the nucleotide sequence of positions 130 to 2232 in SEQ ID NO: 35, the nucleotide sequence of positions 199 to 2100 in SEQ ID NO: 37, the nucleotide sequence of positions 199 to 2325 in SEQ ID NO: 39, the nucleotide sequence of positions 15 to 2325 in SEQ ID NO: 41, the nucleotide sequence of positions 15 to 1916 in SEQ ID NO: 43,
  • polynucleotide sequences of the present invention can be confirmed by using conventional sequence determination methods. For example, the dideoxynucleotide chain termination method (Sanger et al. Proc. Natl. Acad. Sci. USA 1977, 74:5463) can be used. In addition, sequences can also be analyzed using a suitable DNA sequencer.
  • a “protein” in the present invention can also be referred to as a “polypeptide” in general.
  • a “polypeptide” of the present invention refers to a peptide polymer encoded by a polynucleotide of the present invention. Preferred examples include proteins having the amino acid sequence described in SEQ ID NO: 2, 4, 34, 36, 38, 40, 42, 44, 46, 48, or 50.
  • the polypeptides of the present invention may comprise naturally occurring or modified amino acid residues.
  • amino acid residue modifications include acylation, acetylation, amidation, arginylation, GPI anchor formation, crosslinking, ⁇ -carboxylation, cyclization, covalent crosslink formation, glycosylation, oxidation, covalent bonding of a lipid or fat derivative, cystine formation, disulfide bond formation, selenoylation, demethylation, protein fragmentation treatment, covalent bonding of a nucleotide or nucleotide derivative, hydroxylation, pyroglutamate formation, covalent bonding of a flavin, prenylation, covalent bonding with a heme portion, covalent bonding of phosphatidyl inositol, formylation, myristoylation, methylation, ubiquitination, iodination, racemization, ADP-ribosylation, sulfation, and phosphorylation.
  • polypeptides of the present invention include precursors containing a signal peptide portion, mature proteins lacking a signal peptide portion, and fusion proteins modified with other peptide sequences.
  • Peptide sequences to be added to a polypeptide of the present invention can be selected from sequences that facilitate protein purification using, for example, pcDNA3.1/Myc-His vector (Invitrogen), or those that confer stability in recombinant protein production.
  • influenza agglutinin HA
  • glutathione S transferase GST
  • substance P substance P
  • multiple histidine tag such as 6 ⁇ His (SEQ ID NO:60) and 10 ⁇ His (SEQ ID NO:61)
  • protein C fragment HA
  • maltose-binding protein MBP
  • immunoglobulin constant region ⁇ -tubulin fragment
  • ⁇ -tubulin fragment ⁇ -galactosidase, B-tag, c-myc fragment
  • E-tag epipe on a monoclonal phage
  • FLAG Hopp et al. Bio/Technol.
  • lck tag human simple Herpes virus glycoprotein
  • HSV-tag human simple Herpes virus glycoprotein
  • SV40T antigen fragment SV40T antigen fragment
  • T7-tag T7 gene 10 protein
  • VSV-GP fragment vesicular stomatitis virus glycoprotein
  • the GABA-producing neuron progenitor cells of the present invention preferably include, but are not limited to, GABA-producing neuron progenitor cells of the spinal cord and cerebellum.
  • the length of a polynucleotide of the present invention is not particularly limited, as long as it allows for detection or selection of GABA-producing neuron progenitor cells.
  • the polynucleotides of the present invention also include the so-called “oligonucleotides”.
  • the polynucleotides of the present invention comprise at least ten consecutive nucleotides in the nucleotide sequence of the present invention or a complementary sequence thereof, and preferably comprise at least 15 consecutive nucleotides.
  • the present invention also provides nucleotide chains complementary to a polynucleotide for detecting or selecting GABA-producing neuron progenitor cells of the present invention, which comprise at least 15 consecutive nucleotides.
  • Such polynucleotides comprising a nucleotide sequence that contains at least 15 consecutive nucleotides are useful as probes for detecting the generation of GABA-producing neuron progenitor cells or as primers for detecting GABA-producing neuron progenitor cells.
  • the nucleotide chain normally consists of 15 to 100, and preferably 15 to 35 nucleotides and the polynucleotide is appropriately labeled with a radioisotope, non-radioactive compound, or the like when used as a probe.
  • the nucleotide chain preferably consists of at least 15 and preferably 30 nucleotides when used as a primer.
  • a primer can be designed to have a restriction enzyme recognition sequence, a tag or such, added to the 5′-end side thereof, and at the 3′ end, a sequence complementary to a target sequence.
  • a nucleotide chain of the present invention can hybridize with a polynucleotide of the present invention.
  • mutations of a polynucleotide of the present invention within cells can be detected using these probes or primers.
  • such mutations may cause abnormalities in the activity or expression of the polypeptides of the present invention; therefore, nucleotide chains of the present invention are thought to be useful for disease diagnosis, etc.
  • a “complementary sequence” refers to not only cases where at least 15 consecutive nucleotides of the nucleotide sequence completely pair with the template, but also includes those that have at least 70%, preferably 80%, more preferably 90%, and even more preferably 95% or more (for example, 97% or 99%) of the consecutive nucleotides paired with the template.
  • Pair formation refers to the formation of a chain, in which T (U in the case of an RNA) corresponds to A, A corresponds to T or U, G corresponds to C, and C corresponds to G in the nucleotide sequence of the template polynucleotide. Identities can be determined by methods similar to that used in the aforementioned polynucleotide hybridization.
  • the present invention also provides primer sets comprising two or more polynucleotides for detecting or selecting GABA-producing neuron progenitor cells of the present invention.
  • the present invention also provides antibodies that bind to the translation products of the 65B13 gene, which are used in regeneration medicine to treat cerebellar degeneration or spinal cord injury.
  • the present invention provides antibodies for detecting or selecting GABA-producing neuron progenitor cells, which bind to, for example, a protein selected from:
  • “equivalent function to a protein comprising the amino acid sequence of SEQ ID NO: 2, 4, 34, 36, 38, 40, 42, 44, 46, 48, or 50” preferably refers to the selective expression in GABA-producing neuron progenitor cells.
  • the antibodies bind to a polypeptide of the present invention which is an extracellular domain of GABA-producing neuron progenitor cells.
  • the extracellular domains of polypeptides used in the present invention can be searched using the program, PSORT (http://psort.ims.u-tokyo.ac.jp/), etc.
  • extracellular domains obtained using the PSORT program are: the amino acid sequences of positions 21 to 510 in the amino acid sequence of SEQ ID NO: 2, 4, 38, 40, 42, or 44; of positions 20 to 513 in the amino acid sequence of SEQ ID NO: 34 or 36; of positions 21 to 460 in the amino acid sequence of SEQ ID NO: 46 or 48; and of positions 21 to 490 in the amino acid sequence of SEQ ID NO: 50.
  • mutant polypeptide comprising an amino acid sequence, in which one or more amino acids are deleted, inserted, substituted, or added, maintains the same biological activity as the original polypeptide (Mark et al. Proc. Natl. Acad. Sci. USA 1984, 81:5662-6; Zoller and Smith. Nucleic Acids Res. 1982, 10:6487-500; Wang et al. Science 1984, 224:1431-3; Dalbadie-McFarland et al. Proc. Natl. Acad. Sci. USA 1982, 79:6409-13).
  • an amino acid substitution refers to a mutation in which one or more amino acid residues in a sequence are changed to a different type of amino acid residue.
  • a conservative substitution is preferably carried out if the function of the protein is to be maintained.
  • a conservative substitution means altering a sequence so that it encodes an amino acid that has properties similar to those of the amino acid before substitution.
  • Amino acids can be classified, based on their properties, into non-polar amino acids (Ala, Ile, Leu, Met, Phe, Pro, Trp, Val), non-charged amino acids (Asn, Cys, Gln, Gly, Ser, Thr, Tyr), acidic amino acids (Asp, Glu), basic amino acids (Arg, His, Lys), neutral amino acids (Ala, Asn, Cys, Gln, Gly, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val), aliphatic amino acids (Ala, Gly), branched amino acids (Ile, Leu, Val), hydroxyamino acids (Ser, Thr), amide-type amino acids (Gln, Asn), sulfur-containing amino acids (Cys, Met), aromatic amino acids (His, Phe, Trp, Tyr), heterocyclic amino acids (His, Trp), imino acids (Pro, 4Hyp), etc.
  • non-polar amino acids Alky
  • substitutions among Ala, Val, Leu, and Ile; Ser and Thr; Asp and Glu; Asn and Gln; Lys and Arg; and Phe and Tyr are preferable in order to maintain protein properties.
  • a polynucleotide encoding an amino acid sequence, in which one or more amino acids are deleted, inserted, substituted, or added to the amino acid sequence of SEQ ID NO: 2, 4, 34, 36, 38, 40, 42, 44, 46, 48, or 50, can be prepared according to methods such as site-directed mutagenesis described in “Molecular Cloning, A Laboratory Manual 2 nd ed.” (Cold Spring Harbor Press (1989)), “Current Protocols in Molecular Biology” (John Wiley & Sons (1987-1997), Sections 8.1-8.5), Hashimoto-Goto et al. (Gene 1995, 152:271-5), Kunkel (Proc. Natl. Acad. Sci. USA 1985, 82:488-92), Kramer and Fritz (Method. Enzymol. 1987, 154:350-67), Kunkel (Method. Enzymol. 1988, 85:2763-6), etc.
  • proteins of the present invention also include proteins encoded by polynucleotides that hybridize under stringent conditions to a polynucleotide encoding the amino acid sequence of SEQ ID NO: 2, 4, 34, 36, 38, 40, 42, 44, 46, 48, or 50, which are functionally equivalent to a protein comprising the amino acid sequence of SEQ ID NO: 2, 4, 34, 36, 38, 40, 42, 44, 46, 48, or 50.
  • “Equivalent function to a protein comprising the amino acid sequence of SEQ ID NO: 2, 4, 34, 36, 38, 40, 42, 44, 46, 48, or 50” preferably refers to the selective expression in GABA-producing neuron progenitor cells.
  • hybridization conditions in the present invention include “2 ⁇ SSC, 0.1% SDS, 50° C.”, “2 ⁇ SSC, 0.1% SDS, 42° C.”, and “1 ⁇ SSC, 0.1% SDS, 37° C.”.
  • conditions of higher stringency include “2 ⁇ SSC, 0.1% SDS, 65° C.”, “0.5 ⁇ SSC, 0.1% SDS, 42° C.”, and “0.2 ⁇ SSC, 0.1% SDS, 65° C.”. More specifically, a method that uses the Rapid-hyb buffer (Amersham Life Science) can be carried out by performing pre-hybridization at 68° C. for 30 minutes or more, adding a probe to allow hybrid formation at 68° C.
  • conditions of higher stringency can be achieved by increasing the temperature for pre-hybridization, hybridization, or second wash.
  • the pre-hybridization and hybridization temperature can be raised to 60° C., and to 68° C. for higher stringency.
  • a person with ordinary skill in the art can also integrate other conditions such as probe concentration, probe length, and reaction time, to obtain 65B13 isoforms, allelic mutants, and corresponding genes derived from other organisms.
  • hybridizing polynucleotides include polynucleotides containing a nucleotide sequence that has at least 50% or more, preferably 70%, more preferably 80%, and even more preferably 90% (for example, 95% or more, or 99%) identity with a polynucleotide encoding the amino acid sequence of SEQ ID NO: 2, 4, 34, 36, 38, 40, 42, 44, 46, 48, or 50.
  • identities can be determined by the BLAST algorithm (Altschul. Proc. Natl. Acad. Sci. USA 1990, 87:2264-8; Karlin and Altschul. Proc. Natl. Acad. Sci. USA 1993, 90:5873-7).
  • Examples of programs that have been developed based on this algorithm include the BLASTX program for determining the identity of amino acid sequences, and the BLASTN program for nucleotide sequences (Altschul et al. J. Mol. Biol. 1990, 215:403-10). These programs can be used for the sequences of the present invention. One can refer to, for example, http://www.ncbi.nlm.nih.gov for a specific example of analysis methods.
  • the 65B13 isoforms or allelic mutants, and other genes with a 65B13-like structure or function can be obtained from cDNA libraries and genome libraries of animals such as humans, mice, rats, rabbits, hamsters, chickens, pigs, cows, goats, sheep, monkeys, and dogs by designing primers based on the nucleotide sequence of positions 178 to 2280 in SEQ ID NO: 1, the nucleotide sequence of positions 127 to 2079 in SEQ ID NO: 3, the nucleotide sequence of positions 668 to 2770 in SEQ ID NO: 33, the nucleotide sequence of positions 130 to 2232 in SEQ ID NO: 35, the nucleotide sequence of positions 199 to 2100 in SEQ ID NO: 37, the nucleotide sequence of positions 199 to 2325 in SEQ ID NO: 39, the nucleotide sequence of positions 15 to 2325 in SEQ ID NO: 41, the nucleotide sequence of positions 15 to 1916 in SEQ ID NO: 43,
  • Antibodies of the present invention also include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies (scFV) (Huston et al. Proc. Natl. Acad. Sci. USA 1988, 85:5879-83; The Pharmacology of Monoclonal Antibody, vol. 113, Rosenburg and Moore ed., Springer Verlag (1994) pp. 269-315), humanized antibodies, multispecific antibodies (LeDoussal et al. Int. J. Cancer Suppl. 1992, 7:58-62; Paulus. Behring Inst. Mitt. 1985, 78:118-32; Millstein and Cuello. Nature 1983, 305:537-9; Zimmermann Rev. Physiol. Biochem.
  • scFV single-chain antibodies
  • an antibody of the present invention may also be modified by PEG and such, as necessary.
  • An antibody of the present invention may also be produced in the form of a fusion protein with ⁇ -galactosidase, maltose-binding protein, GST, green fluorescent protein (GFP), or such, to allow detection without the use of a secondary antibody.
  • an antibody may be modified by labeling with biotin or such to allow recovery using avidin, streptoavidin, etc.
  • An antibody of the present invention can be produced using a polypeptide of the present invention, a fragment thereof, or a cell in which a polypeptide or polypeptide fragment of the present invention is expressed, as a sensitized antigen.
  • a short polypeptide of the present invention or a fragment thereof may also be used as an immunogen by coupling to a carrier such as bovine serum albumin, Keyhole Limpet Hemocyanin, and ovalbumin.
  • a polypeptide of the present invention or a fragment thereof may be used in combination with a known adjuvant such as aluminum adjuvant, Freund's complete (or incomplete) adjuvant, or pertussis adjuvant, to enhance the immune response to an antigen.
  • Polyclonal antibodies can be obtained from, for example, the serum of an immunized animal after immunizing a mammal with a polypeptide of the present invention or a fragment thereof, together with an adjuvant as necessary.
  • mammals typically include rodents, lagomorphs, and primates. Specific examples include rodents such as mice, rats, and hamsters; lagomorphs such as rabbits; and primates such as monkeys including cynomolgus monkeys, rhesus monkeys, baboons, and chimpanzees.
  • Animal immunization is carried out by suitably diluting and suspending a sensitized antigen in phosphate-buffered saline (PBS) or physiological saline, mixing with an adjuvant as necessary until emulsified, and injecting into an animal intraperitoneally or subcutaneously.
  • PBS phosphate-buffered saline
  • the sensitized antigen mixed with Freund's incomplete adjuvant is preferably administered several times, every 4 to 21 days.
  • Antibody production can be confirmed by measuring the level of an antibody of interest in the serum using conventional methods.
  • the serum itself may be used as a polyclonal antibody, or it may be further purified. See, for example, “Current Protocols in Molecular Biology” (John Wiley & Sons (1987), Sections 11.12-11.13), for specific methods.
  • a monoclonal antibody can be produced by removing the spleen from an animal immunized in the manner described above, separating immunocytes from the spleen, and fusing with a suitable myeloma cell using polyethylene glycol (PEG) or such to establish hybridomas.
  • Cell fusion can be carried out according to the Milstein method (Galfre and Milstein. Methods Enzymol. 1981, 73:3-46).
  • suitable myeloma cells are exemplified particularly by cells that allow chemical selection of fused cells.
  • fused hybridomas are selected by culturing in a culture medium (HAT culture medium) that contains hypoxanthine, aminopterin, and thymidine, which destroy cells other than the fused cells.
  • a clone that produces an antibody against a polypeptide of the present invention or a fragment thereof is selected from the established hybridomas.
  • the selected clone is introduced into the abdominal cavity of a mouse or such, and ascite is collected to obtain a monoclonal antibody. See, in addition, “Current Protocols in Molecular Biology” (John Wiley & Sons (1987), Sections 11.4-11.11) for information on specific methods.
  • Hybridomas can also be obtained by first sensitizing human lymphocytes that have been infected by EB virus with an immunogen in vitro, and fusing the sensitized lymphocytes with human myeloma cells (such as U266) to obtain hybridomas that produce human antibodies (Japanese Patent Application Kokai Publication No. (JP-A) S63-17688 (unexamined, published Japanese patent application)).
  • human antibodies can also be obtained by using antibody-producing cells generated by sensitizing a transgenic animal with a human antibody gene repertoire (WO92/03918; WO93/02227; WO94/02602; WO94/25585; WO96/33735; WO96/34096; Mendez et al. Nat. Genet. 1997, 15:146-156, etc.).
  • Methods that do not use hybridomas can be exemplified by a method in which a cancer gene is introduced to immortalize immunocytes such as antibody-producing lymphocytes.
  • antibodies can also be produced by genetic recombination techniques (see Borrebaeck and Larrick (1990) Therapeutic Monoclonal Antibodies, MacMillan Publishers Ltd., UK).
  • a gene that encodes an antibody is cloned from hybridomas or antibody-producing cells (such as sensitized lymphocytes).
  • the resulting gene is then inserted into a suitable vector, the vector is introduced into a host, and the host is then cultured to produce the antibody.
  • This type of recombinant antibody is also included in the antibodies of the present invention.
  • Typical examples of recombinant antibodies include chimeric antibodies comprising a non-human antibody-derived variable region and a human antibody-derived constant region, and humanized antibodies comprising a non-human-derived antibody complementarity determining region (CDR), human antibody-derived framework region (FR), and human antibody constant region (Jones et al. Nature 1986, 321:522-5; Reichmann et al. Nature 1988, 332: 323-9; Presta. Curr. Op. Struct. Biol. 1992, 2:593-6; Methods Enzymol. 1991, 203:99-121).
  • CDR complementarity determining region
  • FR human antibody-derived framework region
  • Antibody fragments of the present invention can be produced by treating the aforementioned polyclonal or monoclonal antibodies with enzymes such as papain or pepsin.
  • an antibody fragment can be produced by genetic engineering techniques using a gene that encodes an antibody fragment (see Co et al. J. Immunol. 1994, 152:2968-76; Better and Horwitz. Methods Enzymol. 1989, 178:476-96; Pluckthun and Skerra. Methods Enzymol. 1989, 178:497-515; Lamoyi. Methods Enzymol. 1986, 121:652-63; Rousseaux et al. 1986, 121:663-9; Bird and Walker. Trends Biotechnol. 1991, 9:132-7).
  • the multispecific antibodies of the present invention include bispecific antibodies (BsAb), diabodies (Db), etc.
  • Multispecific antibodies can be produced by methods such as (1) chemically coupling antibodies having different specificities with different types of bifunctional linkers (Paulus Behring Inst. Mill. 1985, 78:118-32), (2) fusing hybridomas that secrete different monoclonal antibodies (Millstein and Cuello. Nature 1983, 305:537-9), or (3) transfecting eukaryotic cell expression systems, such as mouse myeloma cells, with a light chain gene and a heavy chain gene of different polyclonal antibodies (four types of DNA), followed by the isolation of a bispecific monovalent portion (Zimmermann. Rev. Physio. Biochem. Pharmacol.
  • diabodies are dimer antibody fragments comprising two bivalent polypeptide chains that can be constructed by gene fusion. They can be produced using known methods (see Holliger et al. Proc. Natl. Acad. Sci. USA 1993, 90:6444-8; EP404097; WO93/11161).
  • Protein A and Protein G can be carried out using Protein A and Protein G, or according to known protein purification techniques (Antibodies: A Laboratory Manual, Ed. Harlow and David Lane, Cold Spring Harbor Laboratory (1988)).
  • Protein A to purify an antibody of the present invention
  • known Protein A columns such as Hyper D, POROS, or Sepharose F.F. (Pharmacia) can be used.
  • the concentration of the resulting antibody can be determined by measuring the absorbance or by enzyme linked immunoadsorbent assay (ELISA).
  • Antigen-binding activity of an antibody can be determined by absorbance measurement, or by using fluorescent antibody methods, enzyme immunoassay (EIA) methods, radioimmunoassay (RIA) methods, or ELISA.
  • EIA enzyme immunoassay
  • RIA radioimmunoassay
  • an antibody of the present invention is first immobilized onto a support such as a plate.
  • a polypeptide of the present invention is added, and then a sample containing the antibody of interest is added.
  • samples containing an antibody of interest include, for example, culture supernatants of antibody-producing cells, purified antibodies, etc.
  • a secondary antibody that recognizes an antibody of the present invention is added, followed by the incubation of the plate. Subsequently, the plate is washed and the label attached to the secondary antibody is detected.
  • the antigen binding activity can be determined by adding an enzyme substrate such as p-nitrophenyl phosphate, and measuring the absorbance.
  • an enzyme substrate such as p-nitrophenyl phosphate
  • a commercially available system such as BIAcore (Pharmacia) can also be used to evaluate antibody activities.
  • the antibodies of the present invention can recognize or detect a polypeptide of the present invention or a fragment thereof. Furthermore, since the antibodies recognize a polypeptide of the present invention or a fragment thereof, they can recognize or detect cells or the like expressing the polypeptide or a fragment thereof. In addition, the antibodies can be used to purify a polypeptide of the present invention or a fragment thereof. The antibodies can also be used to purify cells or the like expressing the polypeptide of the present invention or a fragment thereof.
  • the antibodies of the present invention preferably bind to a polypeptide comprising the entire or at least six consecutive amino acid residues of the amino acid sequence of positions 21 to 510 in the amino acid sequence of SEQ ID NO: 2, 4, 38, 40, 42, or 44; the amino acid sequence of positions 20 to 513 in the amino acid sequence of SEQ ID NO: 34 or 36; the amino acid sequence of positions 21 to 460 in the amino acid sequence of SEQ ID NO: 46 or 48; or the amino acid sequence of positions 21 to 490 in the amino acid sequence of SEQ ID NO: 50, and more preferably bind to a polypeptide comprising at least six consecutive amino acid residues of the above amino acid sequence.
  • the present invention relates to methods for detecting or selecting GABA-producing neuron progenitor cells, which comprise detecting the expression of the above-described polynucleotides of the present invention.
  • the methods of the present invention comprise the step of detecting the expression of a polynucleotide that can hybridize to a polynucleotide of the present invention selected from (i), (ii), (iii), and (iv) described above, or to a complementary sequence thereof.
  • the “step of detecting the expression of a polynucleotide” in the above-described methods of the present invention preferably comprises the steps of:
  • the test cell sample is contacted with a polynucleotide of the present invention or with a probe comprising the polynucleotide.
  • a probe comprising the polynucleotide.
  • the presence of reactivity generally means that the contacted polynucleotide hybridizes (reacts) with the target sequence.
  • steps of the above-described methods of the present invention may include, for example, the steps of:
  • the “gene amplification method” in the above step includes known methods, for example, PCR. Furthermore, the amplification products generated by the amplification method can also be detected by known methods.
  • mRNA prepared from a test cell sample or complementary DNA (cDNA) transcribed from the mRNA may be used as a template in step (a-1) described above.
  • the detection step may be followed by the step of isolating GABA-producing neuron progenitor cells from the detected sample.
  • the protein encoded by the 65B13 gene of the present invention is a membrane protein, viable GABA-producing neuron progenitor cells can be isolated (separate) by using the protein as an indicator.
  • the methods of the present invention may comprise, in addition to the above-described step, the step of detecting or selecting GABA-producing neuron progenitor cells using the expression of a gene selected from the group consisting of Corl1, Pax2, Lim1/2, Lbx1, and Corl2 genes as an indicator.
  • the methods include methods comprising the step of detecting a protein selected from (v), (vi), (vii), and (viii) described above.
  • the protein detection step comprises the steps of:
  • the proteins of the present invention can be detected by contacting an antibody of the present invention with cell samples that may contain GABA-producing neuron progenitor cells, and detecting reactivity.
  • the antibody may be immobilized onto appropriate carriers for use before contact with the cells.
  • cells bound to the antibody can be selectively collected through affinity purification using the antibody after contacting and binding the cells with the antibody.
  • an antibody of the present invention is linked to biotin
  • the cells can be purified by adding the cell sample to a plate or column immobilized with avidin or streptavidin.
  • the detection step may be followed by the step of isolating GABA-producing neuron progenitor cells from the detected sample.
  • GABA-producing neuron progenitor cells can be efficiently separated by flow cytometry using an anti-65B13 antibody.
  • GABA-producing neuron progenitor cells can also be selected using a 65B13 promoter (including modified promoters) (see, for example, JP-A No. 2002-51775).
  • a 65B13 promoter including modified promoters
  • the construct may have a structure where the 65B13 gene is linked upstream or downstream of the marker gene under the control of the expression regulatory sequence (including promoters, enhancers, etc.).
  • the maker gene can be knocked-in at the 65B13 locus.
  • the construct includes, for example, constructs having any one of Structures 2 to 4 schematically illustrated in FIG. 10 .
  • expression of the marker gene is detected specifically in GABA-producing neuron progenitor cells, and this enables specific cell detection.
  • the cell samples used in the methods are culture media containing in vitro differentiated GABA-producing neurons.
  • GABA-producing neurons can be differentiated in vitro by known methods, using known ES cells or the like as a starting material.
  • GABA-producing neurons can be differentiated by co-culturing nerve tissue-derived supporting cell layer with brain tissues obtained from an area containing GABA-producing neurons.
  • the cell sample used for selection of GABA-producing neuron progenitor cells of the present invention may be a group of cells separated or cultured by any method.
  • a support used in immobilizing an antibody or a polypeptide of the present invention is safe to cells.
  • a support include synthetic or naturally occurring organic polymer compounds, inorganic materials such as glass beads, silica gel, alumina, and activated charcoal, and those that have their surfaces coated with a polysaccharide or synthetic polymer.
  • the form of the support examples of which include films, fibers, granules, hollow fibers, non-woven fabric, porous supports, or honeycombed supports, and the contact surface area can be controlled by changing its thickness, surface area, width, length, shape, and size in various ways.
  • Marker proteins for GABA-producing neuron progenitor cells other than the proteins selected by the methods of the present invention include, for example, proteins encoded by genes selected from the group consisting of the Corl1, Pax2, Lim1/2, Lbx1, and Corl2 genes.
  • a transcript of the 65B13 gene can be detected by contacting a polynucleotide of the present invention with nucleic acid extract derived from a cell sample, and detecting for nucleic acid that hybridizes to the polynucleotide in the nucleic acid extract.
  • the polynucleotide probe is preferably labeled with radioisotope or non-radioactive compound to detect a transcript of the 65B13 gene.
  • radioisotopes to be used as a label include, for example, 35 S and 3 H.
  • RNA that binds to a marker can be detected by detecting silver particles by emulsion autoradiography.
  • conventional non-radioisotopic compounds that are used to label polynucleotide probes include biotin and digoxigenin are known.
  • biotin-labeled markers can be achieved, for example, using fluorescent labeled avidin or avidin labeled with an enzyme such as alkaline phosphatase or horseradish peroxidase.
  • the detection of digoxigenin-labeled markers can be achieved by using fluorescent labeled anti-digoxigenin antibody or anti-digoxigenin antibody labeled with an enzyme such as alkaline phosphatase or horseradish peroxidase.
  • enzyme labeling the detection can be made by allowing stable dye to deposit at marker positions by incubating with an enzyme substrate.
  • 65B13 gene transcripts can be detected by amplifying nucleic acid that hybridizes to the polynucleotide primers, for example, using techniques such as RT-PCR.
  • the detection of translation products of the 65B13 gene with the methods of the present invention can be made by contacting the antibody described above with protein extract of cell samples and then detecting proteins bound to the antibody.
  • assay methods for antigen binding activities of antibodies include absorbance measurement, fluorescent antibody method, enzyme immunoassay (EIA), radioimmunoassay (RIA), ELISA, etc.
  • highly accurate identification can be achieved by detecting, in addition to a transcript or translation product of the 65B13 gene, the transcripts or translation products of one or more genes selected from the group consisting of Lbx1, Pax2, Lim1/2, Corl1, and Corl2. Such methods are also included in the present invention.
  • kits for detecting or selecting GABA-producing neuron progenitor cells may comprise, for example, probes, primers, or primer sets that enable detection of the expression of a polynucleotide that can hybridize to a polynucleotide selected from (i), (ii), (iii), and (iv) described above, or to a complementary sequence thereof.
  • the kits may also comprise appropriate buffers, etc.
  • the packages may contain instruction manuals containing a description of how to use the kits.
  • kits of the present invention may further comprise polynucleotides that hybridize to transcripts of one or more genes selected from the group consisting of the Lbx1, Pax2, Lim1/2, Corl1, and Corl2 genes.
  • kits of the present invention may further comprise cerebellar cells or spinal cord cells.
  • kits for detecting or selecting GABA-producing neuron progenitor cells include kits containing antibodies that bind to a protein selected from (v), (vi), (vii), and (viii) described above.
  • kits may further contain in combination antibodies that bind to proteins encoded by one or more genes selected from the group consisting of Lbx1, Pax2, Lim1/2, Corl1, and Corl2 genes.
  • kits may further contain cerebellar cells or spinal cord cells.
  • the preferred target cerebellar cells in the methods or kits of the present invention are Purkinje cells
  • the preferred target spinal cord cells are dI4 and dILA.
  • a polynucleotide of the present invention can be used as an indicator to screen for substances that are effective in differentiating GABA-producing neuron progenitor cells.
  • the present invention provides methods of screening for substances that are effective for differentiating GABA-producing neuron progenitor cells. Since compounds obtained through screening by the methods of the present invention have the activity of differentiating GABA-producing neuron progenitor cells, they are expected to become candidate compounds useful for treating diseases caused by defects in GABA-producing neurons. Target diseases of treatment using a compound obtained by the screening methods include, for example, spinocerebellar ataxia.
  • the above-described screening methods of the present invention comprise the steps of:
  • test substance may be any type of compound, examples of which include the expression products of gene libraries, synthetic low molecular weight compound libraries, synthetic peptide libraries, antibodies, substances released by bacteria, cell (microbial, plant, or animal) extracts, cell (microbial, plant, or animal) culture supernatants, purified or partially purified polypeptides, marine organisms, plant or animal extracts, soil, random phage peptide display libraries, etc.
  • 65B13 is expressed specifically in differentiated GABA-producing neuron progenitor cells, it can be used in screening for reagents that differentiate GABA-producing neuron progenitor cells. Specifically, whether a test sample has the ability to differentiate GABA-producing neuron progenitor cells can be assessed by inducing the differentiation into GABA-producing neuron progenitor cells from cells having the ability to differentiate into GABA-producing neuron progenitor cells in the presence of the test sample, and detecting the expression of 65B13 in the differentiated cells.
  • the present invention provides methods of screening for candidate compounds as a reagent that differentiates GABA-producing neuron progenitor cells, which use as an indicator the expression of 65B13 and comprise the steps of:
  • the preferred “cells having the ability to differentiate into GABA-producing neuron progenitor cells” are cell samples containing cells that can be differentiated into GABA-producing neuron progenitor cells, such as multipotent ES cells.
  • the transcript or translation product of the 65B13 gene of the present invention can be detected using polynucleotides that hybridize to a transcript of the 65B13 gene or antibodies that bind to a translation product of the 65B13 gene, as described above.
  • cell growth and differentiation can be detected by comparing the cell condition with when the test substance is not contacted.
  • Cell growth and differentiation can be assessed through morphological observation under a microscope, or detecting or quantifying substances produced upon cell differentiation.
  • Cell differentiation can be assessed by comparing the expression level of the 65B13 gene with that in the absence of a test sample. Specifically, when a test sample increases the level of transcript or translation product of the 65B13 gene as compared to that in the absence of the test sample, the test sample can be judged to have the ability to differentiate nerve cells. “Increase” means that, for example, the level becomes twice, preferably five times, and more preferably ten or more times.
  • the screening methods of the present invention further comprise the step of selecting compounds with which the expression of the polynucleotide is detected in step (II).
  • the screening methods of the present invention also include, for example, methods comprising the steps of:
  • the above-described methods further comprise step (VI) of selecting compounds with which the protein is detected in step (V).
  • the above methods may comprise use of the 65B13 promoter (including modified promoters) (as a means) to detect proteins.
  • the 65B13 promoter including modified promoters
  • a marker gene can be knocked-in at the 65B13 locus.
  • the expression of the marker gene is detected in a manner specific to GABA-producing neuron progenitor cells, and thus enables detection of the protein.
  • the protein expression can be detected by methods (means) for detecting protein expression, and therefore the methods can also be used as methods (means) for detecting the protein-encoding gene.
  • the gene encoding a marker is linked to a promoter portion means that the gene encoding the marker is linked to the promoter portion in an expressible manner.
  • the gene may be directly linked to the promoter, or the gene may be linked distantly to but still under the control of the promoter.
  • the promoter portion obtained by analyzing the 65B13 expression region may be replaced with another promoter, as long as the promoter for 65B13 enables the expression of the 65B13 region.
  • the present invention also provides methods for producing a cell population of GABA-producing neurons. Such methods include, for example, methods comprising the steps of:
  • the GABA-producing neuron progenitor cells obtained by the production methods described above are used, for example, in treating spinal cord injury or cerebellar degeneration.
  • the GABA-producing neuron progenitor cells obtained by the production methods described above are also included in the present invention. It is preferable that cells produced by the above-described methods of the present invention are viable cells.
  • cells obtained in the present invention are GABA-producing neuron progenitor cells, they are preferable in transplant therapy for degenerative diseases and such in terms of their safety, survival rate, and network formation ability, compared to mixed cell populations or GABA-producing neurons carrying an exogenous gene.
  • cells (or cell populations) of the present invention obtained according to the methods are progenitor cells, they can be differentiated into a suitable stage by selecting in vitro conditions such as media, and are preferable materials for various types of neural transplant therapy.
  • neuron progenitor cells obtained using the methods of the present invention are used in transplants, preferably 1 ⁇ 10 3 to 1 ⁇ 10 6 neurons, and more preferably 5 ⁇ 10 4 to 6 ⁇ 10 4 neurons are transplanted.
  • the primary method is stereotaxic surgery in which a cell suspension is transplanted into the brain.
  • cells may also be transplanted by microsurgery. See, Backlund et al. (J. Neurosurg. 1985, 62:169-73), Lindvall et al. (Ann. Neurol. 1987, 22:457-68), or Madrazo et al. (New Engl. J. Med. 1987, 316:831-4) for methods of transplanting neuron tissues.
  • the cells of the present invention can also be used to isolate genes specific to GABA-producing neuron progenitor cells, and genes specific to each stage of the maturation from progenitor cells into GABA-producing neurons. They can also be used for searching therapeutic targets for degenerative diseases, elucidating the maturation process of GABA-producing neurons, and in screenings using maturation as an indicator.
  • the present invention also provides reagents for detecting or selecting GABA-producing neuron progenitor cells.
  • the representative target cerebellar cell is Purkinje cells and the representative target spinal cord cells are dI4 and dILA.
  • the “cell type identification” means not only when target cells are identified to be of a specific cell type, but also when target cells are judged not to be of a specific cell type.
  • the cells can be identified to be “possibly Purkinje cells” or the cells can be judged “not to be Purkinje cells”.
  • the cells can be identified to be “possibly dI4 or dILA” or the cells can be judged to be “neither dI4 nor dILA”.
  • the reagents of the present invention include, for example, reagents for detecting or selecting GABA-producing neuron progenitor cells, which comprise probes, primers, or primer sets that enable detection of the expression of a polynucleotide that can hybridize to a polynucleotide selected from (i), (ii), (iii), and (iv) described above, or to a complementary sequence thereof.
  • the present invention also provides reagents for detecting or selecting cerebellar cells or spinal cord cells.
  • the above-described reagents for detecting or selecting GABA-producing neuron progenitor cells may be appropriately combined with other known markers.
  • Such reagents enable thorough cell type identification.
  • the present invention provides reagents for detecting or selecting cerebellar cells or spinal cord cells, which comprise a combination of the above-described reagents for detecting or selecting GABA-producing neuron progenitor cells and polynucleotides that hybridize to the transcripts of one or more genes selected from the group consisting of Lbx1, Pax2, Lim1/2, Corl1, and Corl2 genes.
  • the nucleotide sequence of mouse Lbx1 is shown in SEQ ID NO: 5 and the amino acid sequence is shown in SEQ ID NO: 6; the nucleotide sequence of human Lbx1 is shown in SEQ ID NO: 7 and the amino acid sequence is shown in SEQ ID NO: 8.
  • the nucleotide sequence of mouse Pax2 is shown in SEQ ID NO: 9 and the amino acid sequence is shown in SEQ ID NO: 10; the nucleotide sequence of human Pax2 is shown in SEQ ID NO: 11 and the amino acid sequence is shown in SEQ ID NO: 12.
  • the nucleotide sequence of mouse Lim1 is shown in SEQ ID NO: 13 and the amino acid sequence is shown in SEQ ID NO: 14; the nucleotide sequence of human Lim1 is shown in SEQ ID NO: 15 and the amino acid sequence is shown in SEQ ID NO: 16.
  • the nucleotide sequence of mouse Lim2 is shown in SEQ ID NO: 17 and the amino acid sequence is shown in SEQ ID NO: 18; the nucleotide sequence of human Lim2 is shown in SEQ ID NO: 19 and the amino acid sequence is shown in SEQ ID NO: 20; the nucleotide sequence of rat Lim2 is shown in SEQ ID NO: 21 and the amino acid sequence is shown in SEQ ID NO: 22.
  • the nucleotide sequence of mouse Corl1 is shown in SEQ ID NO: 23 and the amino acid sequence is shown in SEQ ID NO: 24; the nucleotide sequence of human Corl1 is shown in SEQ ID NO: 25 and the amino acid sequence is shown in SEQ ID NO: 26; the nucleotide sequence of rat Corl1 is shown in SEQ ID NO: 27 and the amino acid sequence is shown in SEQ ID NO: 28.
  • the nucleotide sequence of mouse Corl2 is shown in SEQ ID NO: 29 and the amino acid sequence is shown in SEQ ID NO: 30; the nucleotide sequence of human Corl2 is shown in SEQ ID NO: 31 and the amino acid sequence is shown in SEQ ID NO: 32.
  • the above-described reagents may further comprise cerebellar cells or spinal cord cells.
  • the reagents of the present invention for detecting or selecting GABA-producing neuron progenitor cells include, for example, reagents for detecting or selecting GABA-producing neuron progenitor cells, which comprise antibodies that bind to a protein selected from (v), (vi), (vii), and (viii) described above.
  • the above-described reagents for detecting or selecting GABA-producing neuron progenitor cells may further comprise in combination antibodies that bind to proteins encoded by one or more genes selected from the group consisting of Lbx1, Pax2, Lim1/2, Corl1, and Corl2 genes.
  • the above-described reagents may further comprise cerebellar cells or spinal cord cells.
  • Cerebellar cells that can be identified using the above-described reagents include Purkinje cells, while spinal cord cells that can be identified using the above-described reagents include dI4 and dILA.
  • 65B13 (Neph3) is selectively expressed in several areas of fetal brain ( FIG. 1 ; WO2004/038018; Minaki Y, Mizuhara E, Morimoto K, Nakatani T, Sakamoto Y, Inoue Y, Satoh K, Imai T, Takai Y, Ono Y. Migrating postmitotic neural precursor cells in the ventricular zone extend apical processes and form adherens junctions near the ventricle in the developing spinal cord. Neurosci Res. 2005, 52(3):250-62). In the midbrain, dopaminergic neuron progenitor cells have been demonstrated to express 65B13 (WO2004/038018); however, the types of cells that express 65B13 remain unidentified in other areas. Thus, the present inventors attempted to identify 65B13-expressing cells in the spinal cord and cerebellar primordia.
  • dI4, dI5, dILA, and dILB form the dorsal horn and transmit sensory, pain, and other signals from the periphery to center.
  • dorsal horn neurons two types (dI4 and dILA) are GABA-producing inhibitory neurons, and the remaining two (dI5 and dILB) are glutamic acid-producing excitatory neurons ( FIG. 2 ).
  • Mouse E10.5 embryos were collected and fixed in 4% PFA/PBS( ⁇ ) at 4° C. for two hours. After replacing with 20% sucrose/PBS( ⁇ ) at 4° C. overnight, the embryos were embedded in OCT. Sections of 12- ⁇ m thickness were prepared and placed onto slide glasses. Then, the sections were dried at room temperature for 30 minutes, and again wetted with PBS( ⁇ ). Next, after 30 minutes of blocking (BlockAce) at room temperature, a primary antibody was reacted at room temperature for one hour. The reaction was followed by incubation at 4° C. overnight.
  • the sections were washed three times with 0.1% Tween-20/PBS( ⁇ ) at room temperature for 15 minutes each, and then incubated with a fluorescently labeled secondary antibody at room temperature for one hour. After washing in the same way, the sections were washed with PBS( ⁇ ) at room temperature for ten minutes, and then mounted.
  • the primary antibodies used were: 65B13 (see WO2004/038018; Minaki Y, Mizuhara E, Morimoto K, Nakatani T, Sakamoto Y, Inoue Y, Satoh K, Imai T, Takai Y, Ono Y. Migrating postmitotic neural precursor cells in the ventricular zone extend apical processes and form adherens junctions near the ventricle in the developing spinal cord. Neurosci Res. 2005, 52(3):250-62), Pax2 (purchased from Zymed), and Mash1 (purchased from BD PharMingen).
  • Corl1 antibody used was described in WO2006/022243 and Mizuhara E, Nakatani T, Minaki Y, Sakamoto Y, Ono Y. Corl1, a novel neuronal lineage-specific transcriptional corepressor for the homeodomain transcription factor Lbx1. J Biol. Chem. 2005, 280(5):3645-55.
  • 65B13 was found to be expressed broadly in the VZ of dorsal spinal cord ( FIG. 1 ).
  • the spatial expression pattern along the dorsoventral axis ( FIG. 4A ) was identical to the pattern of progenitor cell-selective Mash1 expression in dILA and dILB, and the pattern of Corl1 expression in these neurons (Mizuhara E, Nakatani T, Minaki Y, Sakamoto Y, Ono Y. Corl1, a novel neuronal lineage-specific transcriptional corepressor for the homeodomain transcription factor Lbx1. J Biol. Chem. 2005, 280(5):3645-55).
  • 65B13 is selectively expressed in progenitor cells of either or both of dILA and dILB. Unlike at the early stage, two types of neurons develop at this stage in the same area of dorsal spinal cord; therefore, the cell types cannot be identified according to their expression sites. In this context, to identify the cell types, 65B13-positive cells were isolated and cultured using the protocol described below.
  • spinal cord mass was excised from E12.5 mice and dispersed using Cell Suspension Buffer (Invitrogen). Then, without fixation and permeability treatment, the cells were stained at 4° C. for 20 minutes using an anti-65B13 monoclonal antibody (100 times diluted purified antibody, 1% bovine fetal serum, 1 mM EDTA/SDIA differentiation medium (Kawasaki et al. Neuron 2000, 28(1):31-40)). After washing three times with 1 mM EDTA/PBS containing 1% bovine fetal serum at 4° C.
  • the cells were stained with a PE-labeled anti-hamster IgG antibody (Jackson; 10 ⁇ g/ml, 1% bovine fetal serum, 1 mM EDTA/SDIA differentiation medium) at 4° C. for 30 minutes and washed in the same way as described above. After staining, 65B13-expressing cells were separated with a cell sorter. The isolated cells were placed onto slide glasses coated with poly-L-ornithine (Sigma, 0.002% in PBS), laminin (Invitrogen, 5 ⁇ g/ml in PBS), and fibronectin (Sigma, 5 ⁇ g/ml in PBS) and cultured at 37° C.
  • PE-labeled anti-hamster IgG antibody Jackson; 10 ⁇ g/ml, 1% bovine fetal serum, 1 mM EDTA/SDIA differentiation medium
  • 65B13-expressing cells were separated with a cell sorter.
  • the isolated cells were placed onto slide glasses coated with poly-L-
  • SDIA differentiation medium supplemented with Knockout Serum Replacement (Gibco, 5%), N2 (Invitrogen, 1 ⁇ ), B27 (Invitrogen, 1 ⁇ ), ascorbic acid (Sigma, 200 ⁇ M), and BDNF (Invitrogen, 20 ng/ml).
  • the cultured cells were fixed with 2% PFA/PBS at 4° C. for 20 minutes, and then washed twice with PBS at 4° C. for 10 minutes. Then, cell permeability treatment was performed using 0.3% Triton X-100/PBS at room temperature for 30 minutes, and the cells were blocked with 10% normal donkey serum/BlockAce at room temperature for 20 minutes.
  • the cells were incubated with a primary antibody (10% normal donkey serum, 2.5% BlockAce, 0.1% Triton X-100/PBS) at room temperature for one hour and then at 4° C. overnight.
  • a primary antibody (10% normal donkey serum, 2.5% BlockAce, 0.1% Triton X-100/PBS)
  • the cells were incubated with a fluorescently labeled secondary antibodies (all from Jackson, 10 ⁇ g/ml, 10% normal donkey serum, 2.5% BlockAce, 0.1% Triton X-100/PBS) at room temperature for 30 minutes. After washing in the same way as described above, the cells were washed with PBS at room temperature for five minutes, mounted, and observed.
  • the primary antibodies used were: Lim1/2 (purchased from Developmental Studies Hybridoma Bank), HuC/D (purchased from Molecular Probe), and Gad65 (purchased from BD PharMingen).
  • the antibody Lmx1b was prepared by the method described below. First, an expression vector was constructed for a GST fusion protein with amino acids of 271 to 306 of Lmx1b as an immunization antigen. After the resulting vector was introduced into E. coli (JM109 strain), the expression was induced with IPTG. The fusion protein was collected using glutathione beads. After rabbits were immunized several times with the fusion proteins, blood was collected from the rabbits. Anti-Lmx1b polyclonal antibody was obtained by affinity purification of the serum using the same GST-Lmx1b antigen used in the immunization. Nuclear staining was performed using SYTOX Green (Molecular Probe).
  • the first finding was that 65B13-expressing cells in the dorsal spinal cord can be isolated alive by using an anti-65B13 antibody ( FIG. 5A ).
  • Another finding was that almost the entire population of 65B13-expressing cells in the dorsal spinal cord differentiated into HuC/D-positive neurons after two days of culturing, and nearly all of the neurons were Lim1/2-positive and Gad1-positive GABA-producing dILA neurons ( FIG. 5B ).
  • 65B13 was revealed to be selectively expressed in progenitor cells of dILA.
  • 65B13 was specifically expressed in GABA neuron progenitor cells (dI4 and dILA) of the dorsal horn, and these progenitor cells can be separated by using an anti-65B13 antibody.
  • the cerebellum is constituted of glutamic acid-producing granule cells and GABA-producing neurons such as Purkinje cells, Golgi cells, stellate cells, and basket cells (Wang V Y, Zoghbi H Y. Genetic regulation of cerebellar development. Nat Rev Neurosci. 2001, 2(7):484-91).
  • GABA-producing neurons such as Purkinje cells, Golgi cells, stellate cells, and basket cells
  • the spatial expression pattern of 65B13 was compared to those of various markers by the same methods described in Example 1.
  • the anti-Corl2 antibody used was the same as described in WO2006/082826.
  • the cerebellar primordium areas at E12.5 and E14.5 were excised, and 65B13-positive cells were detected and isolated by the same method as described in Example 1 using a cell sorter.
  • the result showed that 65B13-positive cells could be isolated alive from the cerebellar primordium areas at both of the developmental stages ( FIG. 7 ).
  • 65B13 was selectively expressed in progenitor cells of Purkinje and Golgi cells, and these progenitor cells could be separated by using an anti-65B13 antibody. Specifically, 65B13 was demonstrated to be useful as a marker for separating GABA-producing neuron progenitor cells in the spinal dorsal horn and cerebellum.
  • the cell mass was further cultured for four days. Then, the mass was dispersed into cells by the same method as described in Example 1. After staining with the anti-65B13 antibody, the cells were isolated with a cell sorter.
  • 65B13-positive cells were detected in the population of spinal cord cells differentiated from ES cells.
  • 65B13 was demonstrated to be a useful marker for separating not only fetal but also ES cell-derived GABA neuron progenitor cells.
  • the poly A addition sequence of bovine growth hormone (SEQ ID NO: 55; derived from Invitrogen pcDNA3.1+ vector) was amplified and inserted into the HindIII/XhoI site of pSP73 (Promega) to construct pSP73-polyA. Then, the synthetic DNAs of SEQ ID NOs: 56 and 57 were annealed to each other and inserted into the Asp718I/BamHI site of pSP73-polyA to construct pSP73-polyA II.
  • mice genomic fragment (SEQ ID NO: 58) located about 3.2 kb upstream of the translation initiation codon of 65B13 was inserted into the ClaI/Asp718I site of pSP73-polyA II to construct pN3.
  • mouse Gsh1 cDNA (SEQ ID NO: 59) was inserted as a foreign gene into the Asp718I/SalI site of pN3 to construct pN3-Gsh1.
  • pN3-Gsh1 was injected into the pronuclei of mouse fertilized eggs according to the method of Gordon et al. (Gordon J W, Scangos G A, Plotkin D J, Barbosa J A, Ruddle F H.
  • Example 1 Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci USA. 1980 December; 77(12):7380-4), and the eggs were transplanted into foster mothers. The fetuses were recovered at embryonic day 12.5, and the expression of Neph3 and Gsh1 in the cerebellar primordia was analyzed by the methods described in Example 1. An anti-Gsh1 antibody was prepared by the method described below. First, an expression vector was constructed for a GST fusion protein with amino acids of 1 to 72 of Gsh1 as an immunization antigen. After the resulting vector was introduced into E. coli (JM109 strain), the expression was induced with IPTG. The fusion protein was collected using glutathione beads.
  • lymphocytes were collected and fused with myeloma cell P3U1.
  • anti-Gsh1 antibody-producing hybridomas were obtained (hybridoma preparation was outsourced to Kohjin Bio Co.).
  • the present invention identified a selective marker 65B13 for spinal dorsal horn and cerebellar GABA neuron progenitor cells, and successfully isolated GABA neuron progenitor cells by using an antibody against 65B13.
  • This technique can provide viable GABA neuron progenitor cells, and is expected to be useful in preparing materials for transplantation therapy for degenerative diseases, search of specific genes, discovery of drugs targeting GABA neurons, etc.
  • the marker gene identified by the present inventors encodes a membrane protein
  • the marker can be used as an indicator to detect and select GABA neuron progenitor cells and to isolate (separate) viable GABA neuron progenitor cells.
  • Highly pure GABA neurons can be obtained by methods for preparing GABA neuron progenitor cells using the marker of the present invention.
  • the methods are applicable to drug discovery, for example, targeting pain associated with spinal cord GABA neurons, or cerebellar degeneration associated with cerebellar GABA neurons.

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JP5507851B2 (ja) 2014-05-28
US20100303771A1 (en) 2010-12-02
CA2677996A1 (en) 2008-08-14
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